During the past year we have shown that uteroglobin (Utg) is a potent inhibitor of phospholipaseA2 (PLA2) activity in cultured cells as well as in a cell-free system. This suggested that Utg may modulate tissue prostanoids by restricting the level of arachidonate. To study this and the regulation of the Utg gene we have established SV40 ts mutant-transformed cell lines from all organs of the rabbit, known to contain Utg. Two such cell lines derived from the uterine endometrium (RBE-7 and H5DC) have so far been characterized. At 39.4 degrees C stimulated with progesterone (10-9M) the transcription (as measured by dot and Northern blotting) and translation (as measured by Utg RIA) of the Utg gene occur at a rapid rate in these cells. We also found that these cells when treated with phorbol ester, a tumor promoter and an inflamatory agent, increased PLA2 activity by 10-fold with significant increase in tissue prostanoids (PGF2Alpha, PGE2, 6-KetoPFG-1-alpha and TBX2) as measured by HPLC and RIA and compared to control values. When these phorbol ester treated cells were further treated with Utg (20 MuM) 78% reduction in PLA2 activity occured in 12 hours and prostanoid levels decreased 85% of control values. To determine whether endogenous Utg production in these cells would reduce PLA2 activity we treated these cells with progesterone (10-9M) for 24 hours when Utg production is detectable. Significant reduction in PLA2 activity as well as prostanoid levels were observed in these cells. The reduction of PLA2 activity and/or prostanoid level were not observed when either cyclohexamide, a protein synthesis inhibitor, or an antibody to Utg was administered following progesterone treatment. Preliminary in vivo experiments seem to confirm the in vitro results. Additionally, we have successfully subcloned in an expression vector (pKK233-3) the mature Utg coding region from a full-length cDNA containing all the pre-Utg coding sequences. A positive clone with the right orientation of the insert was used to transform two bacterial hosts which have been producing Utg. Site directed mutagenesis studies are now in progress to determine the active site in the Utg molecule involved in PLA2 inhibition.